The present invention relates to a method of imaging cell death in vivo. In particular, it relates to the use of radiolabeled annexin to image regions of cell death in a mammal using gamma ray imaging.
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Apoptotic or programmed cell death plays a crucial role in development and a number of homeostatic and disease processes (Thompson, 1995). New therapeutic strategies of a variety of diseases may therefore be possible through the modulation of apoptotic cell death. The study of new pharmacologic agents to promote or inhibit apoptotic cell death has been impeded by the lack of a noninvasive method(s) to detect and monitor apoptotic cell death in vivo.
Lipid proton nuclear magnetic resonance spectroscopy (1H NMRS) has been found to be useful in the detection of the specific changes of composition and/or fluidity of the plasma membrane of lymphoblasts and other cell lines undergoing apoptotic cell death (Blankenberg, et al., 1996). Clinical use of lipid 1H NMRS study apoptosis is currently limited by complex local magnetic microenvironments found naturally in many tissues and organs.
In one aspect, the present invention includes a method of imaging cell death (e.g., cell death due to apoptosis or necrosis) in a region of a mammalian subject in vivo. The method includes the steps of (a) administering to the subject, annexin coupled, e.g., directly or indirectly, to a biocompatible radionuclide, (b) after a period of time in which the labeled annexin can achieve localization in the subject, positioning the subject within the detection field of a radiation detector device, and (c) measuring radiation emission from the radionuclide localized in the subject, with the radiation detector device, to construct an image of radiation emission, where the image is a representation of cell death in the region of the mammalian subject. In one embodiment, the method further includes a step (d) of processing the image to subtract signal resulting from non-specific localization of the labeled annexin, such as non-specific localization in the kidney.
Radionuclides useful with the method include Iodine 123, Iodine 131, Gallium 67, Indium 111, Fluorine 18, and Technetium 99 m (Tc99m). It will be appreciated that Fluorine 18 is a positron emitter, and is thus useful in positron emission tomography (PET). Iodine 123, Iodine 131, Gallium 67, Indium 111, and Technetium 99 m are useful with standard gamma emission detection. Tc99m is a preferred radionuclide for use with the methods of the invention. In a preferred embodiment, the Tc99m is linked to the annexin via hydrazino nicotinamide (HYNIC). Tc99m-labelled annexin is typically administered at a dose of between about 5 and about 20 mCi.
In one general embodiment of the invention, the radiation detector device is a gamma ray detector device and the measured radiation emission is gamma ray emission. In another general embodiment, the radiation detector device is a positron emission detector device and the measured radiation emission is positron emission.
In yet another general embodiment, the method further includes repeating steps (b) and (c) at selected intervals, where the repeating is effective to track changes in the intensity of radiation emission (e.g., gamma ray or positron emission) from the region over time, reflecting changes in the number of cells undergoing cell death.
Still another general embodiment includes repeating steps (b) and (c) at selected intervals, where the repeating is effective to track changes in the localization of gamma ray emission in the region overtime, reflecting changes in the location of cells undergoing cell death.
The radiation detector device may be, for example, an Anger gamma scintillation camera or a 3-dimensional imaging camera.
A preferred annexin for use with the invention is annexin V. It is typically administered at doses less than about 300 xcexcg protein/kg, preferably between about 1 and 10 xcexcg protein/kg. Several administration routes are possible, including intravenous (i.v.), intraperitoneal (i.p.), intrathecal, and intrapleural administration.
The measuring of gamma ray emission to construct an image is typically done between about 5 minutes and about 2 hours after administration of the labelled annexin. In one embodiment, the measuring of gamma ray emission to construct the image is done about 1 hour after administration of the labelled annexin.
Different portions of the subject may be imaged using the method disclosed herein. For example, the region may include substantially the whole subject, or a portion of the subject, such as the head or portion thereof, the heart or portion thereof, the liver or portion thereof, and the like.
The invention also provides a kit for imaging cell death in vivo. The kit includes (i) a sealed vial containing HYNIC-labeled annexin, prepared, for example, as described in Materials and Methods (A), (ii) a sealed vial containing a Sn-tricine solution prepared, for example, as described in Materials and Methods (B), and maintained under N2, (iii) instructions for making Tc-99m labeled annexin using the components of (I) and (ii) along with Tc-99m, and (iv) instructions for administering the Tc-99m annexin to image areas of cell death in vivo. In one embodiment, the kit is maintained at xe2x88x9270xc2x0 C. and shipped on dry ice. In another embodiment, the HYNIC-labeled annexin is lyophilized.
In another aspect, the present invention provides a composition comprising an annexin, e.g., annexin V, coupled, e.g., directly or indirectly, with a therapeutic radioisotope, e.g., 103Pd, 186Re, 188Re, 90Y, 153Sm, 159Gd, or 166Ho. The therapeutic radioisotope and the annexin may be coupled at a ratio of 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1 (therapeutic radioisotope:annexin). Ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included in the present invention. In one embodiment, the annexin is coupled to a therapeutic radioisotope via a polymeric structure, e.g., dextran or variants thereof.
In a further aspect, the present invention provides a method of tumor radiotherapy by administering to a mammalian subject having a tumor an effective tumor reducing amount of a composition comprising an annexin coupled, e.g., directly or indirectly, with a therapeutic radioisotope. The foregoing method may be used in conjunction with total body irradiation or targeted external irradiation or internal irradiation (e.g., brachytherapy) and/or a treatment employing at least one chemotherapeutic agent (e.g., dimethyl busulfan, cyclophosphamide, bischloroethyl nitrosourea, cytosine arabinoside, or 6-thioguanine). In addition, the method may be used in conjunction with biologically active anti-cancer agents and apoptosis inducing agents such as TNF, TRAIL or Fas or with antibodies, small molecules or pharmacophores which bind these receptors and also induce apoptosis.
In another aspect, the present invention features a method of tumor radiotherapy, which includes treating a subject having a tumor with a chemotherapeutic agent and subsequently administering to the subject an effective tumor reducing amount of a composition comprising an annexin coupled with a therapeutic radioisotope.
The timing of the administration of the annexin coupled with a therapeutic agent is critical to the effectiveness of the therapeutic intervention. The modified annexin should be administered at a time which assures its bioavailability at times of apoptosis or necrosis of the target tissue. Diagnostic imaging studies using radiolabeled annexin V indicate that the administration of a therapeutically modified annexin preferably should be within 24 hours of the completion of a course of chemotherapy with multiple antimetabolite drugs (so-called, CHOP or MOPP therapy) to optimize the availability of annexin localization in the damaged tumor. Optimal time of administration may be within 36, 48, 60, or 72 hours of chemotherapeutic treatment of solid tumors such as breast cancer, lung cancer or sarcoma as shown by imaging studies in patients. Use of a diagnostic imaging agent, such as radiolabeled annexin, to determine the extent of apoptosis may be used to qualify patients for administration of therapeutically modified annexin and to determine the optimal dose of therapeutically modified annexin.
In another aspect, the present invention provides a composition which includes an annexin, e.g., annexin V or a fragment thereof, coupled (directly or indirectly) with a toxin, e.g., E. coli, Salmonella Sp., Listeria Sp., ciguatoxin and related marine polyethers, and aflatoxin.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.